Anti-BDCA2 antibody and its preparation method and use

Abstract

The present disclosure relates to anti-BDCA2 antibodies and methods of making and using the same. In particular, the present disclosure relates to a rabbit-derived antibody that specifically binds to the extracellular region of human BDCA2 and humanized antibodies thereof. The antibodies of the present disclosure have potential therapeutic effects in autoimmune diseases, such as systemic lupus erythematosus.

Description

Technical Field

[0001] The present disclosure relates to the fields of biology and medicine. Specifically, the present disclosure relates to an antibody that specifically binds to human BDCA2, in particular, a rabbit-derived antibody and a humanized antibody thereof that specifically binds to human BDCA2. Background Art

[0002] BDCA2 (Blood dendritic cell antigen 2), also known as CLEC4C or CD303, is the fourth member of the C-type lectin family and a novel type II transmembrane glycoprotein consisting of 213 amino acids. BDCA2 is primarily expressed on the surface of plasmacytoid dendritic cells (pDCs), and its natural ligand has not yet been identified.

[0003] The extracellular portion of BDCA2 contains a C-type carbohydrate recognition domain that can bind to mannose, glucose, or N-acetylglucosamine, effectively inhibiting type I interferon (IFN-I) secretion in pDCs through a mechanism dependent on calcium mobilization and protein tyrosine. Antibodies targeting BDCA2 can rapidly internalize BDCA2 from the pDC surface and reduce the production of inflammatory cytokines, including type I interferons, which play a key role in autoimmune diseases such as systemic lupus erythematosus (SLE). Summary of the Invention

[0004] Antibodies that specifically bind to human BDCA2

[0005] In one aspect, the present disclosure provides an antibody or antigen-binding fragment thereof that specifically binds to human BDCA2, comprising a heavy chain variable region (VH) and a light chain variable region (VL), wherein the heavy chain variable region and the light chain variable region are selected from any one of the following groups:

[0006] The heavy chain variable region comprises HCDR1, HCDR2, and HCDR3 of the sequence shown in SEQ ID NO: 33; the light chain variable region comprises LCDR1, LCDR2, and LCDR3 of the sequence shown in SEQ ID NO: 34; or

[0007] The heavy chain variable region comprises HCDR1, HCDR2, and HCDR3 of the sequence shown in SEQ ID NO: 37; the light chain variable region comprises LCDR1, LCDR2, and LCDR3 of the sequence shown in SEQ ID NO: 38; or

[0008] The heavy chain variable region comprises HCDR1, HCDR2, and HCDR3 of the sequence shown in SEQ ID NO: 31; the light chain variable region comprises LCDR1, LCDR2, and LCDR3 of the sequence shown in SEQ ID NO: 32; or

[0009] The heavy chain variable region comprises HCDR1, HCDR2, and HCDR3 of the sequence shown in SEQ ID NO: 35; the light chain variable region comprises LCDR1, LCDR2, and LCDR3 of the sequence shown in SEQ ID NO: 36; or

[0010] The heavy chain variable region comprises HCDR1, HCDR2, and HCDR3 of the sequence shown in SEQ ID NO: 39; the light chain variable region comprises LCDR1, LCDR2, and LCDR3 of the sequence shown in SEQ ID NO: 40;

[0011] In some embodiments, the HCDRs and the LCDRs are identified according to the Kabat, Chothia, AbM, or IMTG numbering systems.

[0012] In some embodiments, the HCDRs and the LCDRs are identified according to the Kabat numbering system.

[0013] In some embodiments, the antibody or antigen-binding fragment thereof that specifically binds to human BDCA2 as described above comprises a heavy chain variable region and a light chain variable region; the heavy chain variable region and the light chain variable region are selected from any one of the following groups:

[0014] 1) the heavy chain variable region comprises HCDR1 shown in SEQ ID NO: 1, HCDR2 shown in SEQ ID NO: 2, and HCDR3 shown in SEQ ID NO: 3; and

[0015] The light chain variable region comprises LCDR1 shown in SEQ ID NO: 4, LCDR2 shown in SEQ ID NO: 5, and LCDR3 shown in SEQ ID NO: 6; or

[0016] 2) the heavy chain variable region comprises HCDR1 shown in SEQ ID NO: 7, HCDR2 shown in SEQ ID NO: 8, and HCDR3 shown in SEQ ID NO: 9; and

[0017] The light chain variable region comprises LCDR1 shown in SEQ ID NO: 10, LCDR2 shown in SEQ ID NO: 11, and LCDR3 shown in SEQ ID NO: 12; or

[0018] 3) the heavy chain variable region comprises HCDR1 shown in SEQ ID NO: 13, HCDR2 shown in SEQ ID NO: 14, and HCDR3 shown in SEQ ID NO: 15; and

[0019] The light chain variable region comprises LCDR1 shown in SEQ ID NO: 16, LCDR2 shown in SEQ ID NO: 17, and LCDR3 shown in SEQ ID NO: 18; or

[0020] 4) the heavy chain variable region comprises HCDR1 shown in SEQ ID NO: 19, HCDR2 shown in SEQ ID NO: 20, and HCDR3 shown in SEQ ID NO: 21; and

[0021] The light chain variable region comprises LCDR1 shown in SEQ ID NO: 22, LCDR2 shown in SEQ ID NO: 23, and LCDR3 shown in SEQ ID NO: 24; or

[0022] 5) the heavy chain variable region comprises HCDR1 shown in SEQ ID NO: 25, HCDR2 shown in SEQ ID NO: 26, and HCDR3 shown in SEQ ID NO: 27; and

[0023] The light chain variable region comprises LCDR1 shown in SEQ ID NO: 28, LCDR2 shown in SEQ ID NO: 29, and LCDR3 shown in SEQ ID NO: 30;

[0024] The HCDRs and the LCDRs are identified according to the Kabat numbering system.

[0025] In some embodiments, the antibody or antigen-binding fragment thereof that specifically binds to human BDCA2 as described above, wherein the heavy chain variable region and the light chain variable region are selected from any one of the following groups:

[0026] 1) the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 33 or an amino acid sequence having at least 85% sequence identity to SEQ ID NO: 33, and

[0027] the light chain variable region comprises the amino acid sequence set forth in SEQ ID NO:34, or an amino acid sequence that has at least 85% sequence identity to SEQ ID NO:34;

[0028] 2) the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 37 or an amino acid sequence that has at least 85% sequence identity to SEQ ID NO: 37, and

[0029] the light chain variable region comprises the amino acid sequence set forth in SEQ ID NO:38, or an amino acid sequence that has at least 85% sequence identity to SEQ ID NO:38;

[0030] 3) the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 31 or an amino acid sequence having at least 85% sequence identity to SEQ ID NO: 31, and

[0031] the light chain variable region comprises the amino acid sequence of SEQ ID NO:32, or an amino acid sequence that has at least 85% sequence identity to SEQ ID NO:32;

[0032] 4) the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 35 or an amino acid sequence having at least 85% sequence identity to SEQ ID NO: 35, and

[0033] the light chain variable region comprises the amino acid sequence of SEQ ID NO:36, or an amino acid sequence that has at least 85% sequence identity to SEQ ID NO:36;

[0034] 5) the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 39, or an amino acid sequence that has at least 85% sequence identity to SEQ ID NO: 39, and

[0035] the light chain variable region comprises the amino acid sequence of SEQ ID NO:40, or an amino acid sequence that has at least 85% sequence identity to SEQ ID NO:40;

[0036] In this context, "at least 85%" means at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, or a range between any two of the foregoing values, which can be an integer or a decimal.

[0037] In some embodiments, the antibody or antigen-binding fragment thereof that specifically binds to human BDCA2 as described above comprises a heavy chain variable region and a light chain variable region, and the heavy chain variable region and the light chain variable region are selected from any one of the following groups:

[0038] 1) the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 31, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 32;

[0039] 2) the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 33, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 34;

[0040] 3) the amino acid sequence of the heavy chain variable region is set forth in SEQ ID NO: 35, and the amino acid sequence of the light chain variable region is set forth in SEQ ID NO: 36;

[0041] 4) the amino acid sequence of the heavy chain variable region is set forth in SEQ ID NO: 37, and the amino acid sequence of the light chain variable region is set forth in SEQ ID NO: 38;

[0042] 5) the amino acid sequence of the heavy chain variable region is set forth in SEQ ID NO: 39, and the amino acid sequence of the light chain variable region is set forth in SEQ ID NO: 40.

[0043] In some embodiments, the antibody or antigen-binding fragment thereof that specifically binds to human BDCA2 as described in any of the preceding embodiments is a rabbit-derived antibody, a chimeric antibody, or a humanized antibody.

[0044] In some embodiments, the antigen-binding fragment of any of the foregoing antibodies that specifically binds to human BDCA2 is selected from any one of the following: Fab, scFv, Fv, Fab', F(ab')2, single domain antibody, scFab, linear antibody, and multi-specific antibody.

[0045] In some embodiments, the antibody or antigen-binding fragment thereof that specifically binds to human BDCA2 as described in any of the foregoing embodiments further comprises a heavy chain constant region and a light chain constant region.

[0046] In some specific embodiments, the antibody or antigen-binding fragment thereof that specifically binds to human BDCA2 of the present disclosure is rabbit-derived.

[0047] In other specific embodiments, the antibody or antigen-binding fragment thereof that specifically binds to human BDCA2 of the present disclosure is chimeric.

[0048] In other specific embodiments, the antibody or antigen-binding fragment thereof that specifically binds to human BDCA2 of the present disclosure is humanized.

[0049] In some specific embodiments, the antibody or antigen-binding fragment thereof that specifically binds to human BDCA2 of the present disclosure comprises a heavy chain variable region and a light chain variable region, wherein:

[0050] 1) the heavy chain variable region comprises an amino acid sequence selected from any one of SEQ ID NOs: 43-47 or comprises an amino acid sequence having at least 85% sequence identity thereto, and the light chain variable region comprises an amino acid sequence selected from any one of SEQ ID NOs: 48-49 or comprises an amino acid sequence having at least 85% sequence identity thereto; or

[0051] 2) the heavy chain variable region comprises an amino acid sequence selected from any one of SEQ ID NOs: 50 to 52 or an amino acid sequence having at least 85% sequence identity thereto, and the light chain variable region comprises an amino acid sequence selected from any one of SEQ ID NOs: 53 to 54 or an amino acid sequence having at least 85% sequence identity thereto.

[0052] In some particular embodiments, the antibody or antigen-binding fragment thereof of the present disclosure that specifically binds to human BDCA2 comprises a heavy chain variable region and a light chain variable region, wherein:

[0053] 1) the heavy chain variable region comprises an amino acid sequence set forth in SEQ ID NO: 43 or an amino acid sequence having at least 85% sequence identity thereto, and the light chain variable region comprises an amino acid sequence set forth in SEQ ID NO: 48 or an amino acid sequence having at least 85% sequence identity thereto; or

[0054] 2) the heavy chain variable region comprises an amino acid sequence set forth in SEQ ID NO: 44 or an amino acid sequence having at least 85% sequence identity thereto, and the light chain variable region comprises an amino acid sequence set forth in SEQ ID NO: 48 or an amino acid sequence having at least 85% sequence identity thereto; or

[0055] 3) the heavy chain variable region comprises an amino acid sequence set forth in SEQ ID NO: 45 or an amino acid sequence having at least 85% sequence identity thereto, and the light chain variable region comprises an amino acid sequence set forth in SEQ ID NO: 48 or an amino acid sequence having at least 85% sequence identity thereto; or

[0056] 4) the heavy chain variable region comprises an amino acid sequence set forth in SEQ ID NO: 46 or an amino acid sequence having at least 85% sequence identity thereto, and the light chain variable region comprises an amino acid sequence set forth in SEQ ID NO: 48 or an amino acid sequence having at least 85% sequence identity thereto; or

[0057] 5) the heavy chain variable region comprises an amino acid sequence set forth in SEQ ID NO: 47 or an amino acid sequence having at least 85% sequence identity thereto, and the light chain variable region comprises an amino acid sequence set forth in SEQ ID NO: 48 or an amino acid sequence having at least 85% sequence identity thereto; or

[0058] 6) the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 43, or an amino acid sequence having at least 85% sequence identity thereto, and the light chain variable region comprises the amino acid sequence of SEQ ID NO: 49, or an amino acid sequence having at least 85% sequence identity thereto; or

[0059] 7) the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 44, or an amino acid sequence having at least 85% sequence identity thereto, and the light chain variable region comprises the amino acid sequence of SEQ ID NO: 49, or an amino acid sequence having at least 85% sequence identity thereto; or

[0060] 8) the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 45, or an amino acid sequence having at least 85% sequence identity thereto, and the light chain variable region comprises the amino acid sequence of SEQ ID NO: 49, or an amino acid sequence having at least 85% sequence identity thereto; or

[0061] 9) the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 46, or an amino acid sequence having at least 85% sequence identity thereto, and the light chain variable region comprises the amino acid sequence of SEQ ID NO: 49, or an amino acid sequence having at least 85% sequence identity thereto; or

[0062] 10) The heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 47 or an amino acid sequence having at least 85% sequence identity thereto, and the light chain variable region comprises the amino acid sequence shown in SEQ ID NO: 49 or an amino acid sequence having at least 85% sequence identity thereto.

[0063] In some specific embodiments, the antibodies or antigen-binding fragments thereof that specifically bind to human BDCA2 of the present disclosure comprise a heavy chain variable region and a light chain variable region, wherein:

[0064] 1) the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 43, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 48; or

[0065] 2) the amino acid sequence of the heavy chain variable region is as shown in SEQ ID NO: 44, and the amino acid sequence of the light chain variable region is as shown in SEQ ID NO: 48; or

[0066] 3) the amino acid sequence of the heavy chain variable region is set forth in SEQ ID NO:45, and the amino acid sequence of the light chain variable region is set forth in SEQ ID NO:48; or

[0067] 4) the amino acid sequence of the heavy chain variable region is set forth in SEQ ID NO:46, and the amino acid sequence of the light chain variable region is set forth in SEQ ID NO:48; or

[0068] 5) the amino acid sequence of the heavy chain variable region is set forth in SEQ ID NO:47, and the amino acid sequence of the light chain variable region is set forth in SEQ ID NO:48; or

[0069] 6) the amino acid sequence of the heavy chain variable region is set forth in SEQ ID NO:43, and the amino acid sequence of the light chain variable region is set forth in SEQ ID NO:49; or

[0070] 7) the amino acid sequence of the heavy chain variable region is set forth in SEQ ID NO:44, and the amino acid sequence of the light chain variable region is set forth in SEQ ID NO:49; or

[0071] 8) the amino acid sequence of the heavy chain variable region is set forth in SEQ ID NO:45, and the amino acid sequence of the light chain variable region is set forth in SEQ ID NO:49; or

[0072] 9) the amino acid sequence of the heavy chain variable region is set forth in SEQ ID NO:46, and the amino acid sequence of the light chain variable region is set forth in SEQ ID NO:49; or

[0073] 10) the amino acid sequence of the heavy chain variable region is set forth in SEQ ID NO:47, and the amino acid sequence of the light chain variable region is set forth in SEQ ID NO:49.

[0074] In some specific embodiments, the antibodies or antigen-binding fragments thereof of the present disclosure that specifically bind to human BDCA2 comprise a heavy chain variable region and a light chain variable region, wherein:

[0075] 1) the heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO:50, and the light chain variable region comprises the amino acid sequence set forth in SEQ ID NO:53; or

[0076] 2) the heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO:51, and the light chain variable region comprises the amino acid sequence set forth in SEQ ID NO:53; or

[0077] 3) the heavy chain variable region comprises an amino acid sequence as set forth in SEQ ID NO: 52, and the light chain variable region comprises an amino acid sequence as set forth in SEQ ID NO: 53; or

[0078] 4) the heavy chain variable region comprises an amino acid sequence as set forth in SEQ ID NO: 50, and the light chain variable region comprises an amino acid sequence as set forth in SEQ ID NO: 54; or

[0079] 5) the heavy chain variable region comprises an amino acid sequence as set forth in SEQ ID NO: 51, and the light chain variable region comprises an amino acid sequence as set forth in SEQ ID NO: 54; or

[0080] 6) the heavy chain variable region comprises an amino acid sequence as set forth in SEQ ID NO: 52, and the light chain variable region comprises an amino acid sequence as set forth in SEQ ID NO: 54.

[0081] In some specific embodiments, the antibodies or antigen-binding fragments thereof of the present disclosure that specifically bind to human BDCA2 comprise a heavy chain variable region and a light chain variable region, wherein:

[0082] 1) the amino acid sequence of the heavy chain variable region is as set forth in SEQ ID NO: 50, and the amino acid sequence of the light chain variable region is as set forth in SEQ ID NO: 53; or

[0083] 2) the amino acid sequence of the heavy chain variable region is as set forth in SEQ ID NO: 51, and the amino acid sequence of the light chain variable region is as set forth in SEQ ID NO: 53; or

[0084] 3) the amino acid sequence of the heavy chain variable region is as set forth in SEQ ID NO: 52, and the amino acid sequence of the light chain variable region is as set forth in SEQ ID NO: 53; or

[0085] 4) the amino acid sequence of the heavy chain variable region is as set forth in SEQ ID NO: 50, and the amino acid sequence of the light chain variable region is as set forth in SEQ ID NO: 54; or

[0086] 5) the amino acid sequence of the heavy chain variable region is as set forth in SEQ ID NO: 51, and the amino acid sequence of the light chain variable region is as set forth in SEQ ID NO: 54; or

[0087] 6) the amino acid sequence of the heavy chain variable region is as set forth in SEQ ID NO: 52, and the amino acid sequence of the light chain variable region is as set forth in SEQ ID NO: 54.

[0088] In some embodiments, the heavy chain constant region is selected from the constant region of human IgGl, human IgG2, human IgG3, and human IgG4, or a mutant thereof, the mutant having Y at position 252, T at position 254, and E at position 256, or L at position 428 and S at position 434, of the amino acids, the amino acid positions being determined by EU numbering.

[0089] In some embodiments, the light chain constant region is selected from lambda and kappa light chain constant regions.

[0090] In some embodiments, the heavy chain constant region is a heavy chain constant region of human IgGl, and the light chain constant region is a kappa light chain constant region.

[0091] In some embodiments, the heavy chain constant region comprises an amino acid sequence set forth in SEQ ID NO: 41, 60, or 61, and the light chain constant region comprises an amino acid sequence set forth in SEQ ID NO: 42.

[0092] In some specific embodiments, the heavy chain constant region comprises an amino acid sequence of SEQ ID NO: 41, and the light chain constant region comprises an amino acid sequence of SEQ ID NO: 42.

[0093] In some specific embodiments, the heavy chain constant region comprises an amino acid sequence of SEQ ID NO: 41, and the light chain constant region comprises an amino acid sequence of SEQ ID NO: 42.

[0094] In some specific embodiments, the heavy chain constant region is a variant of the amino acid sequence set forth in SEQ ID NO: 41, preferably the heavy chain constant region comprises an amino acid sequence set forth in SEQ ID NO: 60 or 61.

[0095] In some specific embodiments, the antibody or antigen-binding fragment thereof of the present disclosure that specifically binds to human BDCA2 comprises a heavy chain and a light chain, the heavy chain and the light chain being selected from any one of the following groups:

[0096] 1) the heavy chain comprises an amino acid sequence set forth in SEQ ID NO: 55, and the light chain comprises an amino acid sequence set forth in SEQ ID NO: 56;

[0097] 2) the heavy chain comprises an amino acid sequence set forth in SEQ ID NO: 57, and the light chain comprises an amino acid sequence set forth in SEQ ID NO: 58;

[0098] 3) the heavy chain comprises an amino acid sequence set forth in SEQ ID NO: 62, and the light chain comprises an amino acid sequence set forth in SEQ ID NO: 63;

[0099] 4) the heavy chain comprises an amino acid sequence as set forth in SEQ ID NO: 64, and the light chain comprises an amino acid sequence as set forth in SEQ ID NO: 65;

[0100] 5) the heavy chain comprises an amino acid sequence as set forth in SEQ ID NO: 66, and the light chain comprises an amino acid sequence as set forth in SEQ ID NO: 67; and

[0101] 6) the heavy chain comprises an amino acid sequence as set forth in SEQ ID NO: 68, and the light chain comprises an amino acid sequence as set forth in SEQ ID NO: 69.

[0102] In some specific embodiments, the antibody or antigen-binding fragment thereof of the present disclosure that specifically binds to human BDCA2 comprises a heavy chain and a light chain selected from any one of the following groups:

[0103] 1) the heavy chain comprises an amino acid sequence as set forth in SEQ ID NO: 55 or an amino acid sequence having at least 85% sequence identity thereto, and the light chain comprises an amino acid sequence as set forth in SEQ ID NO: 56 or an amino acid sequence having at least 85% sequence identity thereto;

[0104] 2) the heavy chain comprises an amino acid sequence as set forth in SEQ ID NO: 57 or an amino acid sequence having at least 85% sequence identity thereto, and the light chain comprises an amino acid sequence as set forth in SEQ ID NO: 58 or an amino acid sequence having at least 85% sequence identity thereto;

[0105] 3) the heavy chain comprises an amino acid sequence as set forth in SEQ ID NO: 62 or an amino acid sequence having at least 85% sequence identity thereto, and the light chain comprises an amino acid sequence as set forth in SEQ ID NO: 63 or an amino acid sequence having at least 85% sequence identity thereto;

[0106] 4) the heavy chain comprises an amino acid sequence as set forth in SEQ ID NO: 64 or an amino acid sequence having at least 85% sequence identity thereto, and the light chain comprises an amino acid sequence as set forth in SEQ ID NO: 65 or an amino acid sequence having at least 85% sequence identity thereto;

[0107] 5) the heavy chain comprises an amino acid sequence as set forth in SEQ ID NO: 66 or an amino acid sequence having at least 85% sequence identity thereto, and the light chain comprises an amino acid sequence as set forth in SEQ ID NO: 67 or an amino acid sequence having at least 85% sequence identity thereto; and

[0108] 6) the heavy chain comprises an amino acid sequence as set forth in SEQ ID NO: 68 or an amino acid sequence having at least 85% sequence identity thereto, and the light chain comprises an amino acid sequence as set forth in SEQ ID NO: 69 or an amino acid sequence having at least 85% sequence identity thereto.

[0109] In some specific embodiments, the antibody or antigen-binding fragment thereof of the present disclosure that specifically binds to human BDCA2 comprises a heavy chain and a light chain selected from any one of the following groups:

[0110] 1) the amino acid sequence of the heavy chain is as set forth in SEQ ID NO: 55, and the amino acid sequence of the light chain is as set forth in SEQ ID NO: 56;

[0111] 2) the amino acid sequence of the heavy chain is as set forth in SEQ ID NO: 57, and the amino acid sequence of the light chain is as set forth in SEQ ID NO: 58;

[0112] 3) the amino acid sequence of the heavy chain is as set forth in SEQ ID NO: 62, and the amino acid sequence of the light chain is as set forth in SEQ ID NO: 63;

[0113] 4) the amino acid sequence of the heavy chain is as set forth in SEQ ID NO: 64, and the amino acid sequence of the light chain is as set forth in SEQ ID NO: 65;

[0114] 5) the amino acid sequence of the heavy chain is as set forth in SEQ ID NO: 66, and the amino acid sequence of the light chain is as set forth in SEQ ID NO: 67; and

[0115] 6) the amino acid sequence of the heavy chain is as set forth in SEQ ID NO: 68, and the amino acid sequence of the light chain is as set forth in SEQ ID NO: 69.

[0116] In some specific embodiments, the humanized antibody or antigen-binding fragment thereof of the present disclosure binds to human BDCA2 or an extracellular region thereof with a KD value no higher than 4E-09, 3E-09, 2E-09, 1E-09, 9E-10, 8E-10, 7E-10, 6E-10, 5E-10, 4E-10, 3E-10 (M).

[0117] In some specific embodiments, the humanized antibody or antigen-binding fragment thereof of the present disclosure binds to monkey BDCA2 or an extracellular region thereof with a KD value no higher than 7E-09, 6E-09, 5E-09, 4E-09, 3E-09, 2E-09, 1E-09, 9E-10, 8E-10 (M).

[0118] In other specific embodiments, the chimeric antibodies or antigen binding fragments thereof of the present disclosure bind human BDCA2 or an extracellular region thereof with an EC50 of no more than 0.04, 0.03, 0.02, 0.019, 0.018, 0.017, 0.016, 0.015, 0.014, 0.013, 0.012, 0.011 (μg / mL). 50 In other specific embodiments, the chimeric antibodies or antigen binding fragments thereof of the present disclosure bind monkey BDCA2 or an extracellular region thereof with an EC50 of no more than 0.07, 0.06, 0.05, 0.04, 0.03, 0.02 (μg / mL).

[0119] In other specific embodiments, the chimeric antibodies or antigen binding fragments thereof of the present disclosure bind monkey BDCA2 or an extracellular region thereof with an EC50 of no more than 0.07, 0.06, 0.05, 0.04, 0.03, 0.02 (μg / mL). 50 In other specific embodiments, the chimeric antibodies or antigen binding fragments thereof of the present disclosure bind monkey BDCA2 or an extracellular region thereof with an EC50 of no more than 0.07, 0.06, 0.05, 0.04, 0.03, 0.02 (μg / mL).

[0120] In some specific embodiments, the chimeric and humanized antibodies or antigen binding fragments thereof of the present disclosure bind to BDCA2 overexpressing cells.

[0121] The specific binding antibodies or antigen binding fragments thereof to human BDCA2 provided by the present disclosure have any one or more of the following functional properties:

[0122] 1) bind to human or cynomolgus monkey BDCA2 with high binding affinity;

[0123] 2) effectively inhibit TLR9-induced IFN-alpha in human PBMCs;

[0124] 3) effectively inhibit TLR7-induced IFN-alpha in human PBMCs; and

[0125] 4) mediate killing of target cells through ADCC activity.

[0126] In specific embodiments, the specific binding antibodies or antigen binding fragments thereof to human BDCA2 provided by the present disclosure have an EC50 value of 0.0005546 (μg / mL) for inhibition of TLR9-induced IFN-alpha secretion in human PBMCs. The specific testing method can be found in Example 7.

[0127] In specific embodiments, the specific binding antibodies or antigen binding fragments thereof to human BDCA2 provided by the present disclosure have an EC50 value of 0.002841 (μg / mL) for killing of HEK-hBDCA2 cells through ADCC activity, with a maximum killing rate of 38.55%. The specific testing method can be found in Example 9.

[0128] Pharmaceutical composition

[0129] The antibodies or antigen-binding fragments thereof that specifically bind to human BDCA2 according to the present disclosure can be formulated into pharmaceutical compositions for administration to a subject (e.g., to prevent or treat a disease according to the present disclosure). Therefore, the present disclosure provides a pharmaceutical composition comprising a prophylactically or therapeutically effective amount of any of the aforementioned antibodies or antigen-binding fragments thereof that specifically bind to human BDCA2, and one or more pharmaceutically acceptable carriers, diluents, buffers, or excipients.

[0130] Typically, pharmaceutical compositions include a pharmaceutically acceptable carrier, diluent, buffer, or excipient.

[0131] In some embodiments, a "pharmaceutical composition" refers to a mixture containing one or more antibodies or antigen-binding fragments thereof described herein, together with other components, such as physiologically / pharmaceutically acceptable carriers and excipients. One purpose of a pharmaceutical composition is to facilitate administration to an organism, thereby facilitating absorption of the active ingredient and thereby promoting its biological activity.

[0132] In some embodiments, the antibody or antigen-binding fragment thereof described herein is an antibody or antigen-binding fragment thereof that specifically binds to human BDCA2.

[0133] The formulation of drugs is a well-known technique and is further described in, for example, Gennaro (ed.), Remington: The Science and Practice of Pharmacy, 20th ed., Lippincott, Williams & Wilkins (2000); Ansel et al., Pharmaceutical Dosage Forms and Drug Delivery Systems, 7th Ed., Lippincott Williams & Wilkins Publishers (1999).

[0134] Pharmaceutical compositions can also be in a variety of forms, including, for example, liquid, semisolid, and solid dosage forms. For example, liquid solutions (e.g., injectable and infusible solutions), suspensions, tablets, pills, powders, liposomes, suppositories. The preferred form may depend on the mode of administration and therapeutic application. Typically, the pharmaceutical compositions described herein are in the form of injectable or infusible solutions.

[0135] In one embodiment, an antibody that specifically binds to human BDCA2 according to the present disclosure is provided in a buffer at a suitable concentration and stored at 2-8°C.

[0136] The pharmaceutical composition can be administered parenterally (e.g., intravenously, subcutaneously, intraperitoneally, or intramuscularly). As used herein, "parenteral administration" includes, but is not limited to, intravenous, intramuscular, intraarterial, intrathecal, intracapsular, intraorbital, intracardial, intradermal, intraperitoneal, transtracheal, subcutaneous, subcutaneous, intraarticular, subcapsular, or intramuscular administration.

[0137] In some specific embodiments, the antibody or antigen-binding fragment thereof that specifically binds to human BDCA2 is administered intravenously.

[0138] In some embodiments, antibodies or antigen-binding fragments thereof that specifically bind to human BDCA2 can be prepared together with a controlled-release carrier (eg, a controlled-release formulation, an implant, or a microencapsulated delivery system). Methods for preparing such formulations are generally known.

[0139] Isolated nucleic acids

[0140] The present disclosure also provides an isolated nucleic acid encoding any of the aforementioned antibodies or antigen-binding fragments thereof that specifically bind to human BDCA2.

[0141] It should be understood that even though this application provides specific nucleotide sequence examples, the nucleotide sequences capable of "encoding the disclosed antibodies that specifically bind to human BDCA2 or antigen-binding fragments thereof" are not limited to these specific sequences, because equivalent nucleotide sequences can be used to encode the same amino acid sequence depending on the codon preference and codon degeneracy of the expression host.

[0142] expression vector

[0143] The present disclosure also provides an expression vector comprising the isolated nucleic acid according to the present disclosure.

[0144] Nucleic acids encoding the desired antibodies or fragments thereof can be constructed, introduced into expression vectors, and expressed in appropriate host cells.

[0145] The expression vector can be a bacterial plasmid, a bacteriophage, a yeast plasmid, a plant cell virus, a mammalian cell virus or a retrovirus.

[0146] host cells

[0147] The present disclosure also provides a host cell comprising the aforementioned nucleic acid or expression vector for expressing the antibody or antigen-binding fragment thereof that specifically binds to human BDCA2.

[0148] Host cells include prokaryotic and eukaryotic host cells. Eukaryotic host cells include, but are not limited to, mammalian cells, insect cells, plant cells, and fungal cells. Mammalian cells include human, mouse, rat, dog, monkey, pig, goat, cow, horse, and hamster cells. Exemplary host cells include, but are not limited to, CHO, NSO, COS, SP2 cells, HeLa cells, BHK cells, human hepatocellular carcinoma cells, A549 cells, 3T3 cells, and HEK-293 cells. Fungal cells include yeast, such as, but not limited to, Pichia pastoris, Saccharomyces cerevisiae, Hansenula polymorpha, and Kluyveromyces.

[0149] In the present disclosure, a host cell is incapable of developing into a complete animal or plant individual.

[0150] Methods for producing antibodies

[0151] Antibodies can be produced in bacteria or eukaryotic cells. Antibodies can be produced in bacterial cells or in eukaryotic cells (e.g., CHO, 293E). To produce the desired antibody, a nucleic acid encoding the antibody is constructed, introduced into an expression vector, and then expressed in a suitable host cell. Standard molecular biology techniques are used to prepare recombinant expression vectors, transfect host cells, select transformants, culture host cells, and recover the antibody.

[0152] If the antibody is expressed in animal cells (such as CHO), the expression vector includes a promoter required for expression, such as the SV40 promoter, EF1α promoter, or CMV promoter. In addition to the nucleic acid sequence encoding the antibody or its domain, the recombinant expression vector can carry additional sequences, such as a selective marker. The selective marker facilitates the selection of the host cell into which the vector has been introduced. For example, the selection marker is typically resistant to drugs (such as G418, hygromycin, or methotrexate).

[0153] In some embodiments, the antibodies are produced in mammalian cells. Exemplary mammalian host cells for expressing antibodies include Chinese hamster ovary (CHO cells), human embryonic kidney 293 cells, COS cells, NIH3T3 cells, NSO myeloma cells, SP2 cells.

[0154] The antibodies of the present disclosure can be isolated from host cells and purified to substantially pure antibodies. Separation and purification methods commonly used for antibody purification can be used for the separation and purification of antibodies, such as chromatography, filtration, ultrafiltration, salting out, precipitation, extraction, distillation, electrophoresis, isoelectric focusing, dialysis, and recrystallization. Chromatography includes, for example, affinity, ion exchange, hydrophobic, reverse phase, and adsorption chromatography. Columns used for affinity chromatography include, for example, protein A columns and protein G columns.

[0155] In some embodiments, the antibody is produced by chemical synthesis, which includes steps such as synthesizing the amino acid sequence based on the antibody or antigen-binding fragment thereof.

[0156] In some embodiments, the antibodies that specifically bind to human BDCA2 in the present invention include murine antibodies, chimeric antibodies, and humanized antibodies, preferably humanized antibodies.

[0157] Prevention or treatment methods

[0158] The antibodies or antigen-binding fragments thereof that specifically bind to human BDCA2 described herein can be used to treat or prevent various immune diseases, such as inflammatory and autoimmune diseases. In some embodiments, the diseases are associated with abnormal expression or activity of BDCA2 on pDCs.

[0159] The present disclosure provides a method for preventing or treating a disease, comprising administering to a subject a preventive or therapeutically effective amount of an antibody or antigen-binding fragment thereof, nucleic acid, or pharmaceutical composition that specifically binds to human BDCA2. In some embodiments, the disease is an inflammatory disease or an autoimmune disease; more preferably, the inflammatory disease or autoimmune disease is selected from the group consisting of systemic lupus erythematosus, discoid lupus, lupus nephritis, cutaneous lupus erythematosus, rheumatoid arthritis, inflammatory bowel disease, systemic sclerosis, progressive systemic sclerosis (scleroderma), psoriasis, psoriatic arthritis, type I diabetes, fibrosis (e.g., skin fibrosis, pulmonary fibrosis), pemphigus vulgaris, Graves' disease, disease, morphea (localized scleroderma), Sjögren's disease, Hashimoto's disease, dermatomyositis, polymyositis, asthma, Behcet's disease, Crohn's disease, autoimmune glomerulonephritis, membranous glomerulopathy, juvenile rheumatoid arthritis, mixed connective tissue disease, multiple sclerosis, nephrotic syndrome, panniculitis, pemphigoid, pemphigus, pemphigus erythematosus, pemphigus foliaceus, pemphigus vulgaris, polymyalgia rheumatica, Raynaud's phenomenon / syndrome, Sjogren's syndrome, and ulcerative colitis.

[0160] In specific embodiments, the inflammatory or autoimmune disease is systemic lupus erythematosus.

[0161] In specific embodiments, the inflammatory or autoimmune disease is discoid lupus.

[0162] In specific embodiments, the inflammatory or autoimmune disease is cutaneous lupus erythematosus.

[0163] In specific embodiments, the disease or disorder is a BDCA2-mediated disease.

[0164] Systemic lupus erythematosus is a chronic autoimmune disease in which multiple organs are damaged by immune complexes and tissue-bound autoantibodies. Systemic sclerosis (or systemic scleroderma) is a systemic connective tissue disease. Psoriasis is an autoimmune disease that affects the skin. Psoriasis occurs when the immune system mistakes skin cells for pathogens and sends erroneous signals that accelerate the growth cycle of skin cells. Rheumatoid arthritis is a chronic inflammatory disease that affects many tissues and organs, but mainly attacks the movable joints. Inflammatory bowel disease (IBD) is a group of inflammatory diseases of the colon and small intestine. The main types of IBD are Crohn's disease and ulcerative colitis (UC). Dermatomyositis (DM) is a class of autoimmune connective tissue diseases related to polymyositis (PM), characterized by inflammation of the muscles and skin. Type I diabetes is a form of diabetes that results from autoimmune destruction of the insulin-producing β cells of the pancreas, with subsequent insulin deficiency leading to increased blood and urine glucose.

[0165] Examples of other diseases suitable for prevention or treatment using the antibodies or antigen-binding fragments thereof that specifically bind to human BDCA2 include asthma, Behcet's disease, CREST syndrome, pulmonary fibrosis, autoimmune glomerulonephritis, membranous glomerulopathy, juvenile rheumatoid arthritis, mixed connective tissue disease, multiple sclerosis, nephrotic syndrome, panniculitis, pemphigoid, pemphigus, pemphigus erythematosus, pemphigus foliaceus, and pemphigus vulgaris.

[0166] An antibody that specifically binds to human BDCA2, or an antigen-binding fragment thereof, is administered to a subject at risk for, diagnosed with, or experiencing these diseases in an amount and for a period of time to provide a therapeutic effect. The antibody that specifically binds to human BDCA2 (or an antigen-binding fragment thereof) can be administered alone (monotherapy) or in combination with a second therapeutic agent (combination therapy).

[0167] In specific embodiments, the subject is an individual susceptible to, suspected of having, or already having a disease.

[0168] "Treatment" means providing an antibody or antigen-binding fragment thereof that specifically binds to human BDCA2, or a pharmaceutical composition thereof, to a subject experiencing one or more symptoms of a disease. Generally, an antibody or antigen-binding fragment thereof that specifically binds to human BDCA2 is administered to the subject or subjects being treated in an amount effective to alleviate the symptoms of the disease.

[0169] In specific embodiments, the selection of an effective amount can be determined by one skilled in the art based on consideration of a variety of factors (e.g., through clinical trials), including the disease to be treated, the symptoms involved, the route of administration, the severity of the disease, the subject's weight, the subject's immune status, and other factors known to one skilled in the art.

[0170] The effective amount in the specific embodiment can be obtained from the dose-response curve derived from the animal model test system, and is allowed to be determined according to the doctor's judgment and the situation of each subject. As an example, the amount of the drug required for a single administration to a subject can be conveniently obtained by calculating the product of the subject's body weight and the unit weight dose required for the subject's single administration. For example, in the process of preparing a drug, it is generally believed that the adult body weight is 50-70 kg, and the dosage can be initially determined by the equivalent dose conversion relationship between the unit weight dose of the experimental animal and that of a human. For example, it can be determined based on the guidance opinions proposed by drug administration agencies such as the SFDA and the FDA. In some embodiments, the dosage of humans and mice can be converted using a body surface area conversion coefficient of 0.0026 based on humans and mice.

[0171] The active ingredient herein (e.g., an antibody or antigen-binding fragment thereof that specifically binds to human BDCA2) can be administered all at once, or can be divided into a number of smaller unit doses that are administered at intervals. It should be understood that the dosage, duration, and interval of treatment are a function of the disease being treated and can be determined by extrapolation using animal or clinical trial data. Administration can include a single administration or two or more administrations spaced at appropriate intervals. The interval between two adjacent administrations can be 30 minutes, 40 minutes, 50 minutes, 60 minutes, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 12 hours, 14 hours, 16 hours, 18 hours, 20 hours, 22 hours, 24 hours, one and a half days, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months or 12 months.

[0172] The present disclosure provides a method for reducing the production of inflammatory cytokines or chemokines by plasmacytoid dendritic cells in a subject, the method comprising contacting plasmacytoid dendritic cells expressing BDCA2 with an amount of the anti-BDCA2 antibody or antigen-binding fragment thereof, or the pharmaceutical composition of the present disclosure.

[0173] In some embodiments, the use of the antibody or antigen-binding fragment thereof that specifically binds to human BDCA2, or the pharmaceutical composition of the present disclosure, in the manufacture of a medicament for inducing death of plasmacytoid dendritic cells.

[0174] In some embodiments, the use of the antibody or antigen-binding fragment thereof that specifically binds to human BDCA2, or the pharmaceutical composition of the present disclosure, in the manufacture of a medicament for reducing production of inflammatory cytokines or chemokines by plasmacytoid dendritic cells.

[0175] In some embodiments, the antibody or antigen-binding fragment thereof that specifically binds to human BDCA2, or the pharmaceutical composition of the present disclosure, can be used as a medicament for inducing death of plasmacytoid dendritic cells, or reducing production of inflammatory cytokines or chemokines by plasmacytoid dendritic cells.

[0176] use

[0177] In another aspect, the present disclosure also provides the use of the antibody or antigen-binding fragment thereof that specifically binds to human BDCA2, nucleic acid or pharmaceutical composition in the manufacture of a medicament for treating or preventing a disease.

[0178] In some embodiments, the disease is an inflammatory disease or an autoimmune disease.

[0179] In some specific embodiments, the inflammatory disease or autoimmune disease is selected from the group consisting of systemic lupus erythematosus, discoid lupus, lupus nephritis, cutaneous lupus erythematosus, rheumatoid arthritis, inflammatory bowel disease, systemic sclerosis, progressive systemic sclerosis (scleroderma), psoriasis, psoriatic arthritis, type I diabetes, fibrosis (e.g., skin fibrosis, lung fibrosis), pemphigus vulgaris, Graves' disease, scleroderma (localized scleroderma), Sjogren's disease, Hashimoto's disease, dermatomyositis, polymyositis, asthma, Behcet's disease, Crohn's disease, autoimmune glomerulonephritis, membranous glomerulopathy, juvenile rheumatoid arthritis, mixed connective tissue disease, multiple sclerosis, nephrotic syndrome, panniculitis, pemphigoid, pemphigus, pemphigus erythematosus, pemphigus foliaceus, pemphigus vulgaris, polymyalgia rheumatica, Raynaud's phenomenon / syndrome, Sjogren's syndrome, and ulcerative colitis.

[0180] The present disclosure also provides the antibody or antigen-binding fragment thereof that specifically binds to human BDCA2, nucleic acid or pharmaceutical composition for use as a medicament. In some embodiments, the present disclosure also provides the antibody or antigen-binding fragment thereof that specifically binds to human BDCA2, nucleic acid or pharmaceutical composition for use as a medicament for treating a disease.

[0181] In some embodiments, the aforementioned antibody or antigen-binding fragment thereof that specifically binds to human BDCA2 is provided for use in treating a disease, wherein the antibody or antigen-binding fragment thereof that specifically binds to human BDCA2 is administered in combination with a second therapeutic agent.

[0182] In some embodiments, the antibody or antigen-binding fragment thereof that specifically binds to human BDCA2 and the second therapeutic agent are in the same or different containers.

[0183] Detection method

[0184] The antibodies or antigen-binding fragments thereof described herein that specifically bind to human BDCA2 are used in immunoassays. The immunoassay involves contacting the biological sample with the antibodies or antigen-binding fragments thereof described herein to detect the expression of BDCA2 in a biological sample from a subject. Typically, the antibodies or antigen-binding fragments thereof that specifically bind to human BDCA2 described herein can be coupled to a fluorescent label or other label.

[0185] Reagents / Kits

[0186] The present disclosure also provides a drug kit comprising at least one container, wherein each container independently comprises: any one of the aforementioned antibodies or antigen-binding fragments thereof that specifically bind to human BDCA2, nucleic acids, or pharmaceutical compositions.

[0187] In diagnostic or research applications, the kits may comprise one or more of the following components: analytical reagents, buffers, and the antibodies or antigen-binding fragments thereof disclosed herein. In addition, these reagents / kits may comprise instructions.

[0188] The present invention discloses a rabbit-derived antibody, a chimeric antibody thereof, and a humanized antibody thereof that specifically bind to the extracellular region of human BDCA2. These antibodies are found to have high affinity for the BDCA2 protein and have potential therapeutic effects in autoimmune diseases. BRIEF DESCRIPTION OF THE DRAWINGS

[0189] Figure 1 The antibodies disclosed herein inhibit the activity of TLR9-induced IFN-α secretion.

[0190] Figure 2 The antibodies disclosed herein inhibit the activity of TLR7-induced IFN-α secretion.

[0191] Figure 3 . ADCC activity of the antibodies of the present disclosure. DETAILED DESCRIPTION

[0192] the term

[0193] The terms used herein are for the purpose of describing the embodiments only and are not intended to be limiting. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs.

[0194] Unless the context clearly requires otherwise, throughout the specification and claims, the words "comprising," "having," "including," and the like should be understood to mean "including but not limited to." Unless otherwise specified, "comprising" includes "consisting of." For example, for a VH comprising the HCDR1 set forth in SEQ ID NO: 31, it explicitly encompasses the HCDR1 with the amino acid sequence set forth in SEQ ID NO: 31.

[0195] It should be understood that the ordinal numbers "first", "second", "Formula 1", "Formula 2", "1)", "2)", etc. in this disclosure are only used to distinguish different technical features, elements, components, and steps, and are not intended to limit the level, sequence, or quantity.

[0196] A target refers to the object targeted by the therapeutically active ingredients of the present disclosure (antibodies or antigen-binding fragments thereof that specifically bind to human BDCA2). A target can be a nucleic acid (gene, mRNA, etc.) or a protein (precursor, mature protein, isoform, modified form, free form, or surface-expressed). In the present disclosure, a target specifically refers to a protein. As an example, an antibody or antigen-binding fragment thereof that specifically binds to human BDCA2 targets BDCA2.

[0197] The nucleotide or amino acid information of BDCA2 as a target is well known in the art and can be obtained, for example but not limited to, from literature or databases.

[0198] In the context of this disclosure, BDCA2 should be understood broadly to include all forms of BDCA2 molecules at all stages, including, but not limited to, molecules produced during amplification, replication, transcription, splicing, processing, translation, and modification of the BDCA2 gene, such as cDNA, mRNA, precursor protein, mature protein, and fragments thereof. In specific embodiments, BDCA2 refers to human BDCA2. In some specific embodiments, human BDCA2 refers to mature BDCA2 protein, particularly BDCA2 expressed on the cell surface.

[0199] In the field of antibodies, targets typically exist in the form of antigens. "Antigen" refers to a molecule or portion thereof that can be selectively recognized or bound by an antigen-binding molecule (e.g., an antibody or antigen-binding fragment thereof that specifically binds to human BDCA2). An antigen may have one or more epitopes.

[0200] An "epitope" refers to a region on an antigen to which an antibody or antigen-binding fragment thereof specifically binds. An epitope can be formed by contiguous amino acids (linear epitope); or comprise non-contiguous amino acids (conformational epitope). An epitope comprises at least 3, at least 4, at least 5, at least 6, at least 7, or 8-10 amino acids. Antibodies or antigen-binding fragments thereof that bind to a particular epitope can be screened using routine methods in the art, such as, but not limited to, alanine scanning, peptide cleavage analysis, epitope extraction, chemical modification of antigens.

[0201] An "antibody" is used in the broadest sense and encompasses various antibody structures, including but not limited to monoclonal antibodies, polyclonal antibodies, full-length antibodies, as long as they exhibit the desired antigen-binding activity. Typically, a natural IgG antibody is a heterotetrameric protein composed of two light chains and two heavy chains that are linked by disulfide bonds. From N- to C-terminus, each heavy chain has one variable region (VH), three constant domains (CH1, CH2, and CH3). From N- to C-terminus, each light chain has one variable region (VL), one constant light domain (CL). Depending on the context, the skilled artisan can determine the specific meaning of "antibody".

[0202] The "class" of an antibody refers to the type of constant domain or constant region possessed by its heavy chain. Antibodies are classified into five major classes: IgA, IgD, IgE, IgG, and IgM, and several of these can be further divided into subclasses (isotypes), e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2. The heavy chain constant domains that correspond to the different classes of immunoglobulins are called a, d, e, g, and m, respectively.

[0203] "Specifically binds" refers to an antibody or antigen-binding fragment thereof of the present disclosure that binds to the targeted antigen or epitope thereof with higher affinity than to other antigens or epitopes thereof. Typically, the equilibrium dissociation constant (KD) is about 1 x 10 -7 M or less is considered "specific" binding. KD can be measured using known methods, e.g., FACS or surface plasmon resonance. "Specifically binds" does not exclude cross-reactivity to homologous antigens of other species, e.g., Macaca fascicularis, Pan troglodytes, or Callithrix jacchus. In view of this, "specifically binds human BDCA2...", "specifically binds BDCA2..." refers to an antibody or antigen-binding fragment thereof that specifically recognizes or binds to BDCA2 (or an epitope thereof).

[0204] "Affinity" is used to describe the strength of the non-covalent interaction between an antibody or antigen-binding fragment thereof that specifically binds to human BDCA2 and its antigen or epitope. Affinity is typically expressed by the equilibrium dissociation constant (KD). "Kassoc" or "ka" refers to the association rate of a particular antibody-antigen interaction, while "kdis" or "kd" is intended to refer to the dissociation rate of a particular antibody-antigen interaction. "KD" is derived from the ratio of kd to ka (i.e., kd / ka) and is expressed as a molar concentration.

[0205] A "chimeric antibody" is an antibody made by fusing the variable region of an antibody from one species (such as rabbit) with the constant region of an antibody from another species (such as human).

[0206] A "humanized antibody" is an antibody produced by transplanting non-human CDR sequences (such as rabbit-derived CDRs) into the framework of a human antibody variable region.

[0207] "Antibody fragments" or "antigen-binding fragments" are molecules other than intact antibodies that contain a portion of an intact antibody that retains the antigen-binding ability of the intact antibody. Examples of antibody fragments include, but are not limited to, Fv, Fab, Fab', Fab'-SH, F(ab')2, single-domain antibodies, single-chain Fab (scFab), diabodies, linear antibodies, single-chain antibodies (e.g., scFv); and multispecific antibodies formed from antibody fragments.

[0208] "Fab" consists of a light chain and the CH1 and variable region of a heavy chain.

[0209] "Fab' fragment", which is a Fab fragment having one or more cysteine ​​residues at the C-terminus of the CH1 domain.

[0210] "F(ab')2 fragment" is a bivalent fragment formed by two Fab' fragments linked by a disulfide bridge at the hinge region.

[0211] "Fab'-SH" refers to Fab' in which the cysteine ​​residue(s) of the constant domains bear a free thiol group.

[0212] A "single chain Fab fragment" is a polypeptide consisting of an antibody heavy chain variable domain (VH), an antibody constant domain 1 (CH1), an antibody light chain variable domain (VL), an antibody light chain constant domain (CL), and a linker, wherein said antibody domains and said linker have one of the following orders from N- to C-terminal direction: a) VH-CH1-linker-VL-CL, b) VL-CL-linker-VH-CH1, c) VH-CL-linker-VL-CH1, or d) VL-CH1-linker-VH-CL; and wherein said linker is a peptide linker. In some embodiments, the peptide linker is a polypeptide of at least 30 amino acids, preferably a polypeptide of 32-50 amino acids.

[0213] A "diabody" has two antigen binding sites, comprising a heavy chain variable domain (VH) and a light chain variable domain (VL) connected to one another in the same polypeptide chain.

[0214] A "linear antibody" comprises a pair of tandem Fd segments (VH-CH1-VH-CH1) which, together with complementary light chain polypeptides, form a pair of antigen binding regions.

[0215] An "Fv fragment" has the VL and VH domains of one arm of an antibody.

[0216] A "single domain antibody" is an antibody fragment comprising all or a portion of the variable domains of the heavy chains or all or a portion of the variable domains of the light chains of an antibody.

[0217] A single chain antibody fragment (scFv) is an antibody formed by the linkage of a heavy chain variable region and a light chain variable region by a linker.

[0218] An "Fc" contains two heavy chain fragments comprising CH2 and CH3 domains. The two heavy chain fragments are held together by disulfide bonds and by hydrophobic interactions of the CH3 domains. As a supplement, in the anti-BDCA2 antibody or antigen binding fragment thereof of the present application, although the C-terminus of the Fc region is the complete C-terminus ending with PGK, it can also be a truncated C-terminus. Exemplarily, one or two C-terminal residues are removed in the truncated C-terminus (e.g., a truncated C-terminus ending with PG). In a composition comprising antibodies, both antibodies with complete C-termini and antibodies with truncated C-termini can be included.

[0219] A "variable region" refers to the domain in an antibody or antigen binding fragment that binds to an antigen.

[0220] “Complementarity determining region” or “CDR” refers to a region within a variable region that primarily contributes to binding to an antigen. Each of VHand VLcomprises four framework regions (FRs) and three complementarity determining regions (CDRs). VHis comprised of three CDR regions: HCDR1, HCDR2, and HCDR3; VLis comprised of three CDR regions: LCDR1, LCDR2, and LCDR3. Each VHand VLis arranged, from N-terminus to C-terminus, FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4.

[0221] “Framework” or “FR” refers to a variable domain residue other than a hypervariable region (CDR) residue. The FRs of a variable domain typically consist of four FR domains: FR1, FR2, FR3, and FR4. Thus, HVR and FR sequences typically appear in the following order in a VHand (or VL): FR1-H1(L1)-FR2-H2(L2)-FR3-H3(L3)-FR4.

[0222] The heavy chain variable region of an antibody that specifically binds to BDCA2 is denoted as VHand the light chain variable region is denoted as VL. The three CDR regions in VHis denoted as HCDR1, HCDR2, and HCDR3, respectively; the three CDR regions in VLare denoted as LCDR1, LCDR2, and LCDR3, respectively.

[0223] The boundaries of the amino acid sequences of CDRs can be determined by various well-known schemes, such as: the Kabat numbering system (see Kabat et al. (1991), “Sequences of Proteins of Immunological Interest,” 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD), the Chothia numbering system, and the ImMunoGenTics (IMGT) numbering system (Lefranc, M.P. et al., Dev. Comp. Immunol., 27, 55-77 (2003); Front Immunol. 2018 Oct 16;9:2278).

[0224] Correspondence between the various numbering systems is well known to the person skilled in the art. In other words, when a CDR sequence under one numbering system and its position in an antibody are provided, the skilled person is able to determine the corresponding CDR sequence under another numbering system and its position in an antibody. The technical solutions corresponding to the different numbering systems are considered as equivalent technical solutions.

[0225] In one embodiment, the CDRs of the antibodies of the present disclosure are determined positions according to the Kabat numbering scheme.

[0226] The anti-BDCA2 antibodies of the present disclosure can also comprise antibodies having "alternative CDRs." "Alternative" CDRs refer to CDRs (CDR1, CDR2, and CDR3) defined according to any of Chothia (from AbYsis), AbM CDR, or ImMunoGeneTics database (IMGT). These alternative CDRs can be obtained, for example, by using the AbYsis database (www.bioinf.org.uk / abysis / sequence_input / key_annotation / key_annotation.cg). Exemplarily, in the following table, the amino acid sequences of the "alternative" CDR1, CDR2, and CDR3 of the heavy chain variable region and the light chain variable region of 14D10-VH1 VL1 are compared to the CDRs defined according to Kabat.

[0227] Table 1

[0228]

[0229]

[0230] Unless otherwise indicated, in the present disclosure, when referring to residue positions in antibody variable regions and CDRs, the numbering positions refer to the numbering according to the Kabat numbering system.

[0231] In view of the above, the skilled person understands that when describing the antibodies or antigen-binding fragments thereof of the present disclosure that specifically bind to human BDCA2 according to the Kabat numbering system, it also comprises the corresponding sequences under the Chothia, AbM CDR, or IMGT numbering system. As one example, the antibodies or antigen-binding fragments thereof of the present disclosure that specifically bind to human BDCA2, comprise a heavy chain variable region and a light chain variable region; said heavy chain variable region and said light chain variable region are selected from any one of the following groups:

[0232] said heavy chain variable region comprises HCDR1 as set forth in SEQ ID NO: 7, HCDR2 as set forth in SEQ ID NO: 8, HCDR3 as set forth in SEQ ID NO: 9; and

[0233] said light chain variable region comprises LCDR1 as set forth in SEQ ID NO: 10, LCDR2 as set forth in SEQ ID NO: 11, LCDR3 as set forth in SEQ ID NO: 12;

[0234] or

[0235] said heavy chain variable region comprises HCDR1 as set forth in SEQ ID NO: 70, HCDR2 as set forth in SEQ ID NO: 71, HCDR3 as set forth in SEQ ID NO: 9; and

[0236] the heavy chain variable region comprises HCDR1 set forth in SEQ ID NO: 72, HCDR2 set forth in SEQ ID NO: 73, HCDR3 set forth in SEQ ID NO: 9; and

[0237] or

[0238] the heavy chain variable region comprises HCDR1 set forth in SEQ ID NO: 74, HCDR2 set forth in SEQ ID NO: 75, HCDR3 set forth in SEQ ID NO: 9; and

[0239] the heavy chain variable region comprises HCDR1 set forth in SEQ ID NO: 74, HCDR2 set forth in SEQ ID NO: 75, HCDR3 set forth in SEQ ID NO: 9; and

[0240] or

[0241] the heavy chain variable region comprises HCDR1 set forth in SEQ ID NO: 74, HCDR2 set forth in SEQ ID NO: 75, HCDR3 set forth in SEQ ID NO: 9; and

[0242] the heavy chain variable region comprises HCDR1 set forth in SEQ ID NO: 74, HCDR2 set forth in SEQ ID NO: 75, HCDR3 set forth in SEQ ID NO: 9; and

[0243] The term "percent (%) amino acid sequence identity" or simply "identity" is defined as the percentage of amino acid residues in the candidate amino acid sequence that are identical with the amino acid residues in the reference amino acid sequence, after aligning the sequences (and introducing gaps, if necessary) to achieve the maximum percent sequence identity, and not considering any conservative substitutions as part of the sequence identity. Sequence alignment for purposes of determining percent amino acid sequence identity can be achieved using various methods, for example, using the publicly available computer software such as BLAST, BLAST-2, ALIGN, or MEGALIGN (DNASTAR) software. Those skilled in the art can determine appropriate parameters for measuring alignment, including any algorithms needed to achieve maximal alignment over the full length of the sequences being compared.

[0244] As an example, "the heavy chain variable region comprises an amino acid sequence having at least 85% sequence identity to SEQ ID NO: 31" means that the heavy chain variable region comprises HCDR1 set forth in SEQ ID NO: 1, HCDR2 set forth in SEQ ID NO: 2, HCDR3 set forth in SEQ ID NO: 3, and amino acid mutations are allowed in the regions outside of the CDRs so as to have at least 85% sequence identity to SEQ ID NO: 31.

[0245] The term "humanized" refers to a humanized form of a non-human (e.g., murine) antibody or fragment thereof (e.g., Fv, Fab, Fab', F(ab'), scFv, or other antigen-binding portion sequence of an antibody) that contains some portions of sequence derived from a non-human antibody. Humanized antibodies include human immunoglobulins in which residues from a

[0246] Methods for humanizing non-human antibodies are well known in the art. Typically, a humanized antibody has one or more amino acids introduced into it from a non-human source while retaining the original binding activity of the antibody. Methods for humanizing antibodies / antibody molecules are also described in detail in Jones et al., Nature 321 (1986), 522-525; Reichmann et al., Nature 332 (1988), 323-327; and Verhoeyen et al., Science 239 (1988), 1534-1536. A popular method for humanizing antibodies involves CDR grafting, in which a functional antigen binding site from a non-human "donor" antibody is grafted onto a human "acceptor" antibody. CDR grafting methods are known in the art and described in, for example, US 5,225,539, US 5,693,761, and US 6,407,213. Another related method is to produce humanized antibodies from transgenic animals that are genetically engineered to contain one or more humanized immunoglobulin loci capable of gene rearrangement and gene conversion (see, e.g., US 7,129,084).

[0247] An "amino acid mutation" includes an amino acid substitution, deletion, insertion, modification, or any combination thereof. Any combination of substitutions, deletions, insertions and modifications can be made to arrive at the final construct, so long as the final construct possesses the desired properties, such as an amino acid substitution that reduces Fc region homodimerization. Amino acid substitutions can be introduced into an antibody of interest, and the products screened for a desired activity, such as retained / improved antigen binding, reduced immunogenicity. Amino acid sequence deletions and insertions include deletions and insertions at the amino- and / or carboxy-terminus of a polypeptide chain. In one embodiment, the amino acid mutation is a non-conservative amino acid substitution, i.e., the replacement of one amino acid by another amino acid of different structure and / or chemical characteristics. Amino acid mutations can be generated using genetic or chemical methods known in the art. Genetic methods can include site-directed mutagenesis, PCR, gene synthesis, and the like.

[0248] "Nucleic acid" is used interchangeably with "polynucleotide" and refers to deoxyribonucleotides or ribonucleotides and polymers thereof in either single- or double-stranded form. The nucleic acids are synthetic, naturally occurring, and non-naturally occurring. Unless otherwise indicated, nucleic acid sequences also encompass conservatively modified variants (e.g., degenerate substitutions) and complementary sequences, as well as the explicitly indicated sequences. The term "nucleic acid molecule" includes a plurality of nucleic acid molecules.

[0249] "Vector" means a polynucleotide molecule capable of transporting another polynucleotide to which it has been linked. One type of vector is a "plasmid", which refers to a circular double stranded DNA loop into which additional DNA segments can be ligated. Another type of vector is a viral vector, such as an adeno-associated viral vector (AAV), wherein additional DNA segments can be ligated into the viral genome. Some vectors are capable of autonomous replication in a host cell, other vectors can be integrated into the genome of a host after the vector has been introduced into the host so as to be replicated with the host genome.

[0250] "Host cell", "cell line" are used interchangeably and refer to a cell into which exogenous nucleic acid has been introduced and its progeny, without regard to the number of passages. Progeny can not be completely identical to the parental cell both in physical or genetic makeup, but are still within the scope of the term due to mutagenic or other causes. The term "BDCA2-mediated disease" refers to a disease in which BDCA2 is involved in the process of onset, progression or persistence, e.g. involves activation of BDCA2 (e.g. abnormal activation or over-activation), and the involvement of BDCA2 directly or indirectly results in at least one of the following effects: initiation / occurrence of the disease; increase / deepening of pathological changes of the disease; expansion of the site / extent of the disease impact; aggravation of the body damage caused by the disease; increase of the suffering caused by the disease; insensitivity / resistance of the disease to therapeutic measures; decrease of the self-healing tendency of the disease; and deterioration of the prognosis of the disease. In some embodiments of the present disclosure, the BDCA2-mediated disease is an inflammatory disease or an autoimmune disease; in some specific embodiments of the present disclosure, the BDCA2-mediated disease is systemic lupus erythematosus, discoid lupus, lupus nephritis, cutaneous lupus erythematosus, rheumatoid arthritis, inflammatory bowel disease, systemic sclerosis (scleroderma), psoriasis, type I diabetes, fibrosis (e.g. skin fibrosis), pemphigus vulgaris, psoriasis, Graves' disease, scleroderma, Sjogren's disease, Hashimoto's disease, dermatomyositis and polymyositis.

[0251] "Subject" or "individual" refers to an animal, preferably a mammal. According to specific embodiments, the subject is a mammal, including, for example, a camel, a horse, a cow, a pig, a sheep, a cat, a dog, a rabbit, a rat, a guinea pig, a mouse, a primate (e.g. a human). In specific embodiments, the subject is a human.

[0252] It is to be understood that where a numerical range is disclosed herein, such as "A to B", it is meant to include the endpoints of the range and all integers and fractions within the range.

[0253] "and / or", such as "A and / or B" shall be understood to mean "A and B" or "A or B" by itself.

[0254] "Optional" or "optionally" means that the subsequently described event or circumstance can or can not occur, and that the description includes instances where the event or circumstance occurs and instances where it does not. As used in the specification and claims, the singular forms "a," "an" and "the" include their corresponding plural referents unless the context clearly dictates otherwise.

[0255] The following are examples of the practice of this disclosure and should not be interpreted as limiting the scope of the invention in any way.

[0256] Examples

[0257] Example 1. Preparation of rabbit-derived anti-BDCA2 antibodies

[0258] 1. Preparation of immunogen

[0259] The human BDCA2 extracellular region protein with human IgG Fc (hFc) fragment was expressed by Binteng Biotech:

[0260] NFMYSKTVKRLSKLREYQQYHPSLTCVMEGKDIEDWSCCPTPWTSFQSSCYFISTGMQSWTKSQKNCSVMGADLVVINTREEQDFIIQNLKRNSSYFLGLSDPGGRRHWQWVDQTPYNENVTFWHSGEPNNLDERCAIINFRSSEEWGWNDIHCHVPQKSICKMKKIYI (SEQ ID NO: 59); the immunogen hBDCA2-hFc was obtained, with an expression amount of 10.3 mg / L, and a purity of >95% by SDS-PAGE.

[0261] The full-length cDNA sequence of human or monkey BDCA2 (NM_130441.3; XM_005570023.1) and human or monkey FcεRIγ (NM_004106.1; NM_001265840.2) were cloned into lentiviral vector pLVX-Puro (source: Clontech), respectively, 293T cells were transfected by Lipofectamine 2000 (purchased from ThermoFisher; item number: 11668-019), virus supernatant was collected and used to infect target cells, after 2 weeks of screening in selective medium containing puromycin, subcloning was performed in 96-well culture plates by limiting dilution, after 2 weeks, a portion of the monoclonal cells were expanded, and the known anti-BDCA2 antibody 24F4 (produced according to the sequence disclosed in patent application WO2014093396, light / heavy chains corresponding to the third and fourth sequences numbered in WO2014093396) was used for screening by flow cytometry, and the monoclonal cells with higher fluorescence intensity were selected for expansion and cryopreservation, obtaining the overexpression cell strains CHO-K1-HuBDCA2 and CHO-K1-CynoBDCA2 expressing human or monkey BDCA2.

[0262] 2. Animal immunization and antibody screening

[0263] The animals immunized were female New Zealand white rabbits. Rabbit A and B were immunized with hBDCA2-hFc protein as immunogen. The first immunization was emulsified with Freund's complete adjuvant (purchased from Sigma; Cat. No. F5881-10ML) and antigen in equal volume, the dose was 300 μg / rabbit. The booster immunization was emulsified with Freund's incomplete adjuvant (purchased from Sigma, Cat. No. F5506-10ML) and antigen in equal volume, the dose was 150 μg / rabbit. After successful emulsification, the New Zealand white rabbits were injected subcutaneously at multiple sites on the back. The interval between the first immunization and the next was 21 days, and the interval between each immunization was 14 days. The serum was collected after one week of each immunization to test the antibody titer. After the third immunization, ELISA was used to test the serum of rabbit A and B for specific binding to human BDCA2 protein. The results showed that the titer was above 1:256000.

[0264] The CHO-K1-HuBDCA2 stable strain was selected as the immunogen to immunize rabbit C and D. The logarithmic growth phase cells were collected and diluted with PBS, and intraperitoneal immunization was performed at 5x10 7 The interval between the first immunization and the next was 10 days, and the interval between each immunization was one week. After four immunizations, hBDCA2-hFc was used for booster immunization. After the immunization, ELISA was used to test the serum of rabbit C and D for binding to human BDCA2. The results showed that the serum titer of both rabbits was greater than 1:256000.

[0265] After the immunization, the B cells of the animals were taken and specifically screened with immunomagnetic beads. The B cell complete culture medium was added, and the cells were plated in 96-well cell culture plates by limited dilution. After 7-10 days of culture, ELISA and FACS were performed for detection. A total of 355 positive B cell supernatants were screened, and 42 positive clones were selected for sequencing and expression.

[0266] Example 2. Sequencing of light / heavy chain variable regions and expression of chimeric antibodies

[0267] The positive B cells cultured in Example 1 were collected for RNA extraction and reverse transcription. After sequencing, the heavy chain and light chain variable region sequences of the positive clones were obtained, and rabbit anti-4C12, 14D10, 14G5, 16C6, and 17H5 were obtained. The CDR amino acid sequences were determined according to the database of Kabat (Kabat, E. A. et al. 1991).

[0268] The sequenced heavy and light chain variable regions were ligated with the human antibody heavy chain IgG1 constant region and the human antibody light chain kappa constant region, respectively, and cloned into the expression vector pcDNA3.4. CHO-K1 cells were transiently transfected and cultured at 37°C, 8% CO2, and 110 rpm for 3 to 7 days. Cell supernatants were collected and purified by protein A affinity chromatography to obtain chimeric antibodies ch-4C12, ch-14D10, ch-14G5, ch-16C6, and ch-17H5. The purified antibodies were analyzed for protein concentration, purity, endotoxin detection, and various characterization tests. Five antibodies were ultimately obtained, whose CDRs and heavy and light chain variable region amino acid sequences are shown in the table below.

[0269] Table 2. CDR sequences of rabbit antibodies

[0270]

[0271] Note: The CDRs in the table are obtained according to the Kabat numbering convention.

[0272] The variable regions of the rabbit antibody are as follows:

[0273] 4C12-VH:

[0274] QSLEESGGRLVTPGTPLTLTCTVSGFSLN NYAVS WVRQAPGKGLEWIG LIS NSGNTYYASWAKG RFTISKTSTTTVDLKGTSPTTEDTATYFCAR YGYDDYGYY WLDL WGQGTLVTVSS (SEQ ID NO: 31)

[0275] 4C12-VL:

[0276] IKMTQTPASVSAAVGGTVSINC QASEDIESYLA WYQQKPGQPPKLLIY DAD DLAS GVPSRFKGSGSGTQFTLTISGVQCDDAATYYC QHGHYTSSSDNA FGGGT EVVVK (SEQ ID NO: 32)

[0277] 14D10-VH:

[0278] QSVEESGGRLVTPGTPLTLTCTVSGIDLS TYGMG WVRQAPGKGLEYIG IISN TGGTYSASWAKG RFTISKTSTTTVDLKMTSLTTEDTATYFCAR GLYDDYGTYYF DL WGQGTLVTVSS(SEQ ID NO:33)

[0279] 14D10-VL:

[0280] IKMTQTPASVSAAVGGRVSINC QASEDIESYLA WYQQKPGQPPKLLIY DAS DLAS GVPSRFKGSGSGKQFTLTISGVQCDDAATYYC QHGFYTSDSDNA FGGGT EVVVE(SEQ ID NO:34)

[0281] 14G5-VH:

[0282] QSVEESGGRLVTPGTPLTLTCTVSGFSLS EYSMG WVRQAPGKGLEYIG IISN TGNTYYASWAKG RFTISKTSTTTVDLRTTSPTTEDTATYFCAR SLYDDYGAYYF DL WGQGTLVTVSS(SEQ ID NO:35)

[0283] 14G5-VL:

[0284] IKLTQTPASVSAAVGGTVTINC QASEDIETYLA WYQQKPGQPPNLLIY DAS DLAS GVPSRFKGSGSGKQFTLTISGVQCDDAATYYC QHGYYTSTSDNA FGGGT AVVVK(SEQ ID NO:36)

[0285] 16C6-VH:

[0286] QSVEESGGRLVTPGTPLTLTCTVSGFSLS SYAMS WVRQAPGKGLEWIG IISS SGNTYYASWAKG RFAISKTSTKVDLRITSPTTEDTATYFCAR GYSYDDYGDFFD AFDP WGPGTLVTVSS(SEQ ID NO:37)

[0287] 16C6-VL:

[0288] ADIVMTQTPASVEAAVGGTVTIKC QASQSIVGYLA WYQQKPGQPPKLLIY RASTLPS GVPSRFKGSGSGAQFTLTISDLECADAATYYC QSWHYDTSSTYGWA F GGGTEVVVK(SEQ ID NO:38)

[0289] 17H5-VH:

[0290] QSVEESGGRLVTPGTPLTLTCTVSGFSLS SYAMS WVRQAPGKGLEWIG IIST SGRTYDASWAKG RFTISKTSTTVDLKITSPTTEDTATYFCAR ALYDDYGDYWG YFNL WGQGTLVTVSS(SEQ ID NO:39)

[0291] 17H5-VL:

[0292] IKMTQTPASVSAAVGGTVSINC QASEDIETFLA WYQQKPGQPPKLLIY DAS DLAS GVPSRFKGSGSGKQFTLTISGVQCDDAATYYC QHGYYTSSSENA FGGGT EVVVK(SEQ ID NO:40);

[0293] Human IgGl constant region:

[0294] ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 41)

[0295] Human light chain kappa constant region:

[0296] RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGN SQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRG EC (SEQ ID NO: 42).

[0297] Example 3. Humanization of the antibodies

[0298] The germline gene sequences with the highest homology to the rabbit antibody heavy chain variable region and light chain variable region were selected as the variable region grafting scaffolds by sequence alignment.

[0299] 14D10 selected IGHV3-64, IGHV3-23 and IGHV1-46 as the heavy chain variable region grafting scaffold, and selected IGKV1-5 and IGKV3-7 as the light chain variable region grafting scaffold. 16C6 selected IGHV3-64 as the heavy chain variable region grafting scaffold, and selected IGKV1-27 and IGKV1-12 as the light chain variable region grafting scaffold. The grafted scaffold regions were back mutated by homology modeling, and the humanized antibodies of 14D10 and 16C6 were designed and expressed.

[0300] The variable region sequences of the humanized antibodies are as follows:

[0301] 14D10-VH1:

[0302] QVQLVESGGGLVQPGGSLRLSCSASGIDLSTYGMGWVRQAPGKGLEYIGII SNTGGTYSASWAKGRFTISKDSSKNTVYLQMNSLRAEDTAVYYCARGLYDDY GTYYFDLWGRGTLVTVSS (SEQ ID NO: 43)

[0303] 14D10-VH2:

[0304] QVQLVESGGGLVQPGGSLRLSCSASGIDLSTYGMGWVRQAPGKGLEYIGII SNTGGTYSASWAKGRFTISKDSSKNTLYLQMNSLRAEDTAVYYCARGLYDDY GTYYFDLWGRGTLVTVSS (SEQ ID NO: 44)

[0305] 14D10-VH3:

[0306] QVQLVESGGGLVQPGGSLRLSCSASGIDLSTYGMGWVRQAPGKGLEYIGII SNTGGTYSASWAKGRFTISKDNSKNTLYLQMNSLRAEDTAVYYCARGLYDDYGTYYFDLWGRGTLVTVSS(SEQ ID NO:45)

[0307] 14D10-VH7:

[0308] EVQLLESGGGLVQPGGSLRLSCAASGIDLSTYGMGWVRQAPGKGLEYIGIISNTGGTYSASWAKGRFTISKDSSKNTVYLQMNSLRAEDTAVYYCARGLYDDYGTYYFDLWGRGTLVTVSS(SEQ ID NO:46)

[0309] 14D10-VH8:

[0310] QVQLVQSGAEVKKPGASVKVSCKASGIDLSTYGMGWVRQAPGQGLEYIGIISNTGGTYSASWAKGRFTITKDSSTSTVYMELSSLRSEDTAVYYCARGLYDDYGTYYFDLWGRGTLVTVSS(SEQ ID NO:47)

[0311] 14D10-VL1:

[0312] DIQMTQSPSSVSASVGDRVTITCQASEDIESYLAWYQQKPGKAPKLLIYDASDLASGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQHGFYTSDSDNAFGGGTKVEIK(SEQ ID NO:48)

[0313] 14D10-VL2:

[0314] EIVMTQSPPTLSLSPGERVTLSCQASEDIESYLAWYQQKPGQAPRLLIYDASDLASGIPARFSGSGSGKDFTLTISSLQPEDFAVYYCQHGFYTSDSDNAFGGGTKVEIK(SEQ ID NO:49)

[0315] 16C6-VH1:

[0316] QVQLVESGGGLVQPGGSLRLSCSASGFSLSSYAMSWVRQAPGKGLEWISIISSSGNTYYASWAKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARGYSYDDYGDFFDAFDPWGQGTMVTVSS (SEQ ID NO: 50)

[0317] 16C6-VH2:

[0318] QVQLVESGGGLVQPGGSLRLSCSASGFSLSSYAMSWVRQAPGKGLEWIGIISSSGNTYYASWAKGRFTISKDSSKNTVYLQMNSLRAEDTAVYYCARGYSYDDYGDFFDAFDPWGQGTMVTVSS (SEQ ID NO: 51)

[0319] 16C6-VH3:

[0320] QVQLVESGGGLVQPGGSLRLSCSASGFSLSSYAMSWVRQAPGKGLEWIGIISSSGNTYYASWAKGRFTISKDSSKNTVYLQMNSLRAEDTAVYYCARGYSYDDYGDFFDAFEPWGQGTMVTVSS (SEQ ID NO: 52)

[0321] 16C6-VL1:

[0322] DIQMTQSPSSLSASVGDRVTITCQASQSIVGYLAWYQQKPGKVPKLLIYRASTLPSGVPSRFSGSGSGTDFTLTISSLQPEDVATYYCQSWHYDTSSTYGWAFGQGTKVEIK (SEQ ID NO: 53)

[0323] 16C6-VL2:

[0324] DIQMTQSPSSVSASVGDRVTITCQASQSIVGYLAWYQQKPGKAPKLLIYRASTLPSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQSWHYDTSSTYGWAFGQGTKVEIK (SEQ ID NO: 54).

[0325] The heavy chain and light chain variable regions of the humanized antibody were cloned into the expression vector pcDNA3.4, respectively, and linked to the human antibody heavy chain IgGl constant region and the human antibody light chain kappa constant region, respectively, to obtain the humanized antibody by transient expression according to the method of Example 2.

[0326] Table 3. Sequence combinations of the variable regions of the humanized antibodies

[0327]

[0328] The sequences of exemplary humanized antibodies are as follows:

[0329] 14D10-VH1VL1 HC:

[0330] QVQLVESGGGLVQPGGSLRLSCSASGIDLSTYGMGWVRQAPGKGLEYIGIISNTGGTYSASWAKGRFT ISKDSSKNTVYLQMNSLRAEDTAVYYCARGLYDDYGTYYFDLWGRGTLVTVSS ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 55)

[0331] 14D10-VH1VL1 LC:

[0332] DIQMTQSPSSSVSASVGDRVTITCQASEDIESYLAWYQQKPGKAPKLLIYDASDLASGVPSRFSGSGSG TDFTLTISSLQPEDFATYYCQHGFYTSDSDNAFGGGTKVEIK RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 56)

[0333] 16C6-VH1VL1 HC:

[0334] QVQLVESGGGLVQPGGSLRLSCSASGFSLSSYAMSWVRQAPGKGLEWISIISSSGNTYYASWAKGRFT ISRDNSKNTLYLQMNSLRAEDTAVYYCARGYSYDDYGDFFDAFDPWGQGTMVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPA VLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPC PAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVH NAKTKPREEQYNSTYRWSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKG QPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPV LDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 57)

[0335] 16C6-VH1 VL1 LC:

[0336] DIQMTQSPSSSLSASVGDRVTITCQASQSIVGYLAWYQQKPGKVPKLLIYRASTLPSGVPSRFSGSGSG TDFTLTISSLQPEDVATYYCQSWHYDTSSTYGWAFGQGTKVEIK RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDST YSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 58).

[0337] Underlined indicates variable region.

[0338] Example 4. Fc mutations of humanized antibodies

[0339] The heavy chain constant region of the humanized antibody screened in Example 3 was introduced with M252Y, S254T, and T256E (YTE) mutations, or introduced with M428L and N434S (LS) mutations to increase the affinity of the antibody to FcRn. The amino acid positions are determined by EU numbering.

[0340] Fc-YTE:

[0341] Bold is the mutation site;

[0342] Fc-LS:

[0343] Bold is the mutation site.

[0344] The sequences of exemplary mutant antibodies are as follows:

[0345] Heavy chain sequence of 14D10-VH1 VL1 (YTE):

[0346]

[0347] Light chain sequence of 14D10-VH1 VL1 (YTE):

[0348] DIQMTQSPSSSVSASVGDRVTITCQASEDIESYLAWYQQKPGKAPKLLIYDASDLASGVPSRFSGSGSG TDFTLTISSLQPEDFATYYCQHGFYTSDSDNAFGGGTKVEIK RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 63; same as 14D10-VH1 VL1 LC)

[0349] Heavy chain sequence of 14D10-VH1 VL1 (LS):

[0350]

[0351] Light chain sequence of 14D10-VH1 VL1 (LS):

[0352] DIQMTQSPSSSVSASVGDRVTITCQASEDIESYLAWYQQKPGKAPKLLIYDASDLASGVPSRFSGSGSG TDFTLTISSLQPEDFATYYCQHGFYTSDSDNAFGGGTKVEIK RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 65; same as 14D10-VH1 VL1 LC)

[0353] Heavy chain sequence of 16C6-VH1 VL1 (YTE):

[0354]

[0355] Light chain sequence of 16C6-VH1 VL1 (YTE):

[0356] DIQMTQSPSSSLSASVGDRVTITCQASQSIVGYLAWYQQKPGKVPKLLIYRASTLPSGVPSRFSGSGSG TDFTLTISSLQPEDVATYYCQSWHYDTSSTYGWAFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 67, same as 16C6-VH1VL1 LC)

[0357] Heavy chain sequence of 16C6-VH1VL1(LS):

[0358]

[0359] Light chain sequence of 16C6-VH1VL1(LS):

[0360] DIQMTQSPSSSLSASVGDRVTITCQASQSIVGYLAWYQQKPGKVPKLLIYRASTLPSGVPSRFSGSGSG TDFTLTISSLQPEDVATYYCQSWHYDTSSTYGWAFGQGTKVEIK RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 69, the same as 16C6-VH1VL1 LC);

[0361] The mutation sites are in bold and the variable regions are underlined.

[0362] Example 5. Antibody Binding Activity Detection

[0363] 1. Antibody affinity testing

[0364] Octet R8 was used to detect the affinity of the antibody to BDCA2 protein.

[0365] Sample Preparation: Dilute the test antibody to 5 μg / mL using SD running buffer (0.1% BSA-PBST). The analyte, human BDCA2 protein (purchased from ACRO; Catalog No. CLC-H5245) or monkey BDCA2 (purchased from KACTUS; Catalog No. BCA-CM102), was diluted to 5 μg / mL and then further diluted two-fold to a total of five concentrations. A ProA biosensor (purchased from Sartorius) was used to capture the antibody, and the sensor was immersed in the analyte.

[0366] This example ran five steps: 1. Baseline (60 seconds), 2. Loading (capture antibody) (100 seconds, 1.0 nm), 3. Baseline (60 seconds), 4. Association (45 seconds), and 5. Dissociation (200 seconds). After completion of the experiment, Octet Analysis Studio 12.2 software was used for fitting. The results are shown in the table below.

[0367] The results showed that all the screened chimeric antibodies had high affinity to BDCA2, and the chimeric and humanized antibodies of 14D10 and 16C6 had high affinity to both human and cynomolgus monkey BDCA2.

[0368] Table 4. Affinity of chimeric antibodies to hBDCA2

[0369] Antibody KD(M) kon(1 / Ms) kdis(1 / s) ch-4C12 7.34E-11 4.67E+05 3.43E-05 ch-14D10 2.96E-10 3.09E+05 9.16E-05 ch-14G5 3.03E-10 3.09E+05 9.34E-05 ch-16C6 1.03E-09 2.44E+05 2.51E-04 ch-17H5 3.41E-10 3.38E+05 1.15E-04

[0370] Table 5. Affinity of 14D10 humanized antibody to BDCA2 from different species

[0371]

[0372] Table 6. Affinity of 16C6 humanized antibody to BDCA2 from different species

[0373]

[0374]

[0375] 2. ELISA to detect the binding of antibodies to BDCA2 protein

[0376] The binding of antibodies to human or monkey BDCA2 protein was detected by ELISA.

[0377] Human BDCA2 (purchased from Kactus; Catalog No.: BCA-HM102) or monkey BDCA2 (purchased from Kactus; Catalog No.: BCA-CM102) was diluted in PBS at a concentration of 1 μg / mL and coated on a 96-well plate overnight at 4°C. The plate was blocked with 5% BSA-PBST blocking buffer at 37°C for 1 hour. After washing, serially diluted antibodies were added (20 μg / mL in the first well, followed by 6-fold serial dilutions) and incubated at 37°C for 1 hour. After washing, HRP-conjugated goat anti-human IgG (purchased from Sigma; Catalog No.: 129825) was added and incubated at 37°C for 30 minutes. After washing, TMB was added and color was developed in the dark for 10 minutes. The reaction was then terminated with 2N H2SO4. The absorbance was measured at 450 nm on a microplate reader (TECAN SPARK).

[0378] The results showed that the chimeric antibodies had good binding activity to both human and monkey BDCA2, and the binding activity of the humanized antibodies 14D10 and 16C6 to hBDCA2 protein was comparable to that of the chimeric antibodies.

[0379] Table 7. Binding activity of chimeric antibodies to hBDCA2 and cynoBDCA2

[0380]

[0381] Table 8. Binding activity of antibodies to hBDCA2

[0382]

[0383]

[0384] 3. Detection of Antibody Binding Activity to Cells Expressing BDCA2

[0385] Flow cytometry (FACS) was used to detect the binding of humanized antibodies to cells overexpressing BDCA2.

[0386] The 293T-hBDCA2 cells in Example 1 were collected and washed twice with PBS containing 2% BSA. 5 Cells were added to a 96-well cell culture plate, and antibodies at different dilutions were added (80 μg / mL in the first well, 4-fold serial dilutions), and incubated on ice for 30 minutes. After washing, PE-labeled goat anti-human IgG (purchased from Invitrogen; catalog number: 12-4998-82) was added, and the cells were incubated at 4°C in the dark for 30 minutes. Flow cytometry (BD Accuri C6 Plus) was used for detection.

[0387] The results showed that the humanized antibodies and chimeric antibodies of 14D10 and 16C6 had comparable binding activities to 293T-hBDCA2 cells.

[0388] Table 9. Antibody binding activity to 293T-hBDCA2 cells

[0389]

[0390] Example 6. Detection of Antibody Endocytosis Ability

[0391] The HEK293 cells expressing human BDCA2 in Example 1 were collected and cultured at a volume of 5×10 3Cells were seeded into 96-well white microplates at 1 μg / well and cultured overnight in an incubator. Antibodies were mixed with DT3C (purchased from Huamei Biotechnology; Catalog No. CSB-EP360556CQR1) at a molar ratio of 1:4 and incubated at room temperature for 30 minutes. The antibody-DT3 mixture was diluted to 3 μg / mL in the first well, followed by 2.5-fold serial dilutions before addition to the cells. Cultured in a 37°C, 5% CO2 incubator for 3 days, CTG (purchased from Promega; Catalog No. G7572) was then added for detection.

[0392] The results showed that the chimeric antibodies all had good endocytosis activity; the binding activities of 14D10 and 16C6 humanized antibodies were comparable to those of the chimeric antibodies.

[0393] Table 10. Endocytic activity of chimeric antibodies

[0394]

[0395] Example 7. Antibodies inhibit TLR9-induced IFN-α secretion

[0396] Human PBMC cells were resuspended in complete culture medium (RPMI 1640 + 10% FBS + 1×NEAA + 1× sodium pyruvate + 1×GlutaMax) and the cell density was adjusted to 5×10 6 / mL, inoculated into a 96-well cell culture plate at 100μL / well; diluted the antibody to 40μg / mL, diluted 20-fold to 2 concentration points, diluted 10-fold to 1 concentration point, and then diluted 3-fold to 4 concentration points, and added to the plate at 50μL / well; ODN2216 (manufacturer: Invivogen; catalog number: tlrl-2216-1) was diluted to 2μM, added to the plate at 50μL / well, and cultured at 37°C for 18 hours. The supernatant was collected and the IFN-α concentration in the supernatant was detected using an IFN-α ELISA kit (purchased from Dayou; catalog number: 1110013). The results are shown in Table 11 and Figure 1 As shown, the humanized antibody can significantly inhibit the secretion of IFN-α induced by TLR9.

[0397] Table 11. Antibody inhibition of IFN-α secretion activity

[0398] Antibody EC50 (μg / mL) 14D10-VH1VL1 0.0005546

[0399] Example 8. Antibodies inhibit TLR7-induced IFN-α secretion

[0400] Human PBMC cells were resuspended in complete culture medium (RPMI 1640 + 10% FBS + 1×NEAA + 1× sodium pyruvate + 1×GlutaMax) and the cell density was adjusted to 5×10 6 / mL, inoculated into a 96-well cell culture plate at 100μL / well; diluted the antibody to 40μg / mL, diluted 20-fold to 2 concentration points, diluted 10-fold to 1 concentration point, and then diluted 3-fold to 4 concentration points, and added to the plate at 50μL / well; added 50μL of 4μg / ml concentration of R837 (manufacturer: Invivogen; catalog number: tlrl-imqs) and cultured at 37℃ for 18 hours. The supernatant was collected and the IFN-α concentration in the supernatant was detected using an IFN-α ELISA kit (purchased from Dayou; catalog number: 1110013). The results are shown in Tables 12 and Figure 2 As shown, the humanized antibodies had a significant inhibitory effect on the secretion of IFN-α induced by TLR7.

[0401] Table 12. Antibody inhibition of IFN-α secretion activity

[0402] Antibody EC50 (μg / mL) 14D10-VH1VL1 0.0005933

[0403] Example 9. Antibody ADCC activity detection

[0404] PBMCs were used to detect the ADCC activity of humanized antibodies. PBMCs were revived and cultured at a rate of 2×10 5 HEK-hBDCA2 cells were collected and the cell density was adjusted to 1×10 4 The antibody was diluted to a concentration of 2 μg / mL in the first well, and then added to the plate after a 7-fold serial dilution. The plate was incubated at 37°C for 6 h. The LDH kit (purchased from Roche; manufacturer: 04744934001) was used for color development, and the plate was read on a microplate reader.

[0405] The results are shown in Table 13 and Figure 3 As shown, 14D10-VH1VL1 mediated the killing of target cells through ADCC.

[0406] Table 13. Antibody ADCC activity

[0407] Antibody EC50 (μg / mL) Maximum kill rate (%) 24F4 0.004632 30.15 14D10-VH1VL1 0.002841 38.55

Claims

1. An antibody or antigen-binding fragment thereof that specifically binds to human BDCA2, comprising a heavy chain variable region and a light chain variable region; wherein: The heavy chain variable region comprises HCDR1 shown in SEQ ID NO: 7, HCDR2 shown in SEQ ID NO: 8, and HCDR3 shown in SEQ ID NO: 9; and The light chain variable region comprises LCDR1 shown in SEQ ID NO: 10, LCDR2 shown in SEQ ID NO: 11, and LCDR3 shown in SEQ ID NO: 12; The amino acid sequences of the HCDR and the LCDR are determined according to the Kabat numbering system.

2. An antibody or antigen-binding fragment thereof that specifically binds to human BDCA2, comprising a heavy chain variable region and a light chain variable region; wherein: The heavy chain variable region comprises HCDR1 shown in SEQ ID NO: 70, HCDR2 shown in SEQ ID NO: 71, and HCDR3 shown in SEQ ID NO: 9; and The light chain variable region comprises LCDR1 shown in SEQ ID NO: 10, LCDR2 shown in SEQ ID NO: 11, and LCDR3 shown in SEQ ID NO: 12; The amino acid sequences of the HCDR and the LCDR are determined according to the Chothia numbering system.

3. An antibody or antigen-binding fragment thereof that specifically binds to human BDCA2, comprising a heavy chain variable region and a light chain variable region; wherein: The heavy chain variable region comprises HCDR1 shown in SEQ ID NO: 72, HCDR2 shown in SEQ ID NO: 73, and HCDR3 shown in SEQ ID NO: 9; and The light chain variable region comprises LCDR1 shown in SEQ ID NO: 10, LCDR2 shown in SEQ ID NO: 11, and LCDR3 shown in SEQ ID NO: 12; The amino acid sequences of the HCDR and the LCDR are determined according to the AbM numbering system.

4. An antibody or antigen-binding fragment thereof that specifically binds to human BDCA2, comprising a heavy chain variable region and a light chain variable region; wherein: The heavy chain variable region comprises HCDR1 shown in SEQ ID NO: 74, HCDR2 shown in SEQ ID NO: 75, and HCDR3 shown in SEQ ID NO: 9; and The light chain variable region comprises LCDR1 shown in SEQ ID NO: 76, LCDR2 shown in SEQ ID NO: 77, and LCDR3 shown in SEQ ID NO: 12; The amino acid sequences of the HCDR and the LCDR are determined according to the IMGT numbering system. 5 . The antibody or antigen-binding fragment thereof that specifically binds to human BDCA2 according to claim 1 , which is a rabbit-derived antibody or antigen-binding fragment thereof, a chimeric antibody or antigen-binding fragment thereof, or a humanized antibody or antigen-binding fragment thereof.

6. The antibody or antigen-binding fragment thereof that specifically binds to human BDCA2 according to any one of claims 1 to 4, wherein: The amino acid sequence of the heavy chain variable region is as shown in SEQ ID NO: 33 or has at least 85% sequence identity to SEQ ID NO: 33, and The amino acid sequence of the light chain variable region is as shown in SEQ ID NO: 34 or has at least 85% sequence identity to SEQ ID NO:

34.

7. The antibody or antigen-binding fragment thereof that specifically binds to human BDCA2 according to any one of claims 1 to 4, wherein the antigen-binding fragment is selected from any one of the following: Fab, scFv, Fv, Fab', F(ab')2, scFab, linear antibody, and multispecific antibody.

8. The antibody or antigen-binding fragment thereof that specifically binds to human BDCA2 according to any one of claims 1 to 4, which is a humanized antibody; The amino acid sequence of the heavy chain variable region is selected from any one of SEQ ID NOs: 43 to 47 or has at least 85% sequence identity thereto, and The amino acid sequence of the light chain variable region is selected from any one of SEQ ID NOs: 48 to 49 or has at least 85% sequence identity thereto. 9 . The antibody or antigen-binding fragment thereof that specifically binds to human BDCA2 according to claim 1 , further comprising a heavy chain constant region and a light chain constant region.

10. The antibody or antigen-binding fragment thereof that specifically binds to human BDCA2 according to claim 9, wherein the heavy chain constant region is selected from the constant regions of human IgG1, human IgG2, human IgG3, and human IgG4, or mutants thereof, wherein the mutant of the heavy chain constant region of human IgG1 has the amino acid at position 252 mutated to Y, the amino acid at position 254 mutated to T, and the amino acid at position 256 mutated to E, or the amino acid at position 428 mutated to L, and the amino acid at position 434 mutated to S, wherein the amino acid positions are determined by EU numbering; The light chain constant region is selected from a lambda light chain constant region and a kappa light chain constant region.

11. The antibody or antigen-binding fragment thereof that specifically binds to human BDCA2 according to claim 9, wherein: The heavy chain constant region comprises the amino acid sequence shown in SEQ ID NO: 41, 60 or 61, The light chain constant region comprises the amino acid sequence shown in SEQ ID NO:

42.

12. The antibody or antigen-binding fragment thereof that specifically binds to human BDCA2 according to any one of claims 1 to 4, comprising a heavy chain and a light chain, wherein the heavy chain and the light chain are selected from any one of the following groups: 1) the amino acid sequence of the heavy chain is as shown in SEQ ID NO: 55 or has at least 85% sequence identity thereto, and The amino acid sequence of the light chain is as shown in SEQ ID NO: 56 or has at least 85% sequence identity thereto; 2) the amino acid sequence of the heavy chain is SEQ ID NO: 62 or has at least 85% sequence identity thereto, and The amino acid sequence of the light chain is as set forth in SEQ ID NO: 63 or has at least 85% sequence identity thereto; 3) the amino acid sequence of the heavy chain is SEQ ID NO: 64 or has at least 85% sequence identity thereto, and The amino acid sequence of the light chain is as shown in SEQ ID NO: 65 or has at least 85% sequence identity thereto.

13. A pharmaceutical composition comprising: 1) a therapeutically effective amount of an antibody or antigen-binding fragment thereof that specifically binds to human BDCA2 according to any one of claims 1 to 12, and 2) One or more pharmaceutically acceptable carriers, diluents, buffers or excipients.

14. A biomaterial comprising: 1) A nucleic acid molecule encoding the antibody or antigen-binding fragment thereof that specifically binds to human BDCA2 according to any one of claims 1 to 12; 2) A vector comprising the nucleic acid molecule described in 1) above; 3) A cell comprising the nucleic acid molecule of 1) above or the vector of 2) above.

15. Use of the antibody or antigen-binding fragment thereof that specifically binds to human BDCA2 according to any one of claims 1 to 12, or the pharmaceutical composition according to claim 13, in the preparation of a medicament for treating and / or preventing a disease, wherein the disease is systemic lupus erythematosus or cutaneous lupus erythematosus.

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